Genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in Belgium.

Borrelia burgdorferi sensu lato (s.l.), the etiological agent of Lyme disease, is transmitted by the bite of Ixodes ricinus. Four hundred eighty-nine ticks, collected in four locations of a region of southern Belgium where Lyme disease is endemic, were examined for the presence of the spirochete. In a PCR test with primers that recognize a chromosomal gene of all strains, 23% of the ticks were found to be infected. The species B. burgdorferi s.l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s.s.), Borrelia garinii, and Borrelia afzelii, which could be distinguished in PCR tests with species-specific primers that correspond to distinct plasmid sequences. B. garinii was most prevalent (53% of infected ticks), followed by B. burgdorferi s.s. (38%) and B. afzelii (9%). Of the infected ticks, 40% were infected with a single species, 40% were infected with two species, and 5% were infected with all three species. For 15% of the ticks, the infecting species could not be identified. No difference in rates of prevalence was observed among the four locations, which had similar ground covers, even though they belonged to distinct biogeographic regions. A greater heterogeneity of spirochetal DNA in ticks than in cultured reference DNA was suggested by a comparison of the results of PCRs with two different sets of species-specific primer sequences.

Species-specific plasmid sequences for PCR identification of the three species of Borrelia burgdorferi sensu lato involved in Lyme disease.

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

Cloning and sequencing of a species-specific nucleotide fragment of Borrelia burgdorferi sensu stricto, which is repeated in several plasmids of the species.

Among the etiological agents of Lyme disease, Borrelia burgdorferi sensu stricto strains carry a 16 kb plasmid, which did not hybridize to plasmids of B. garinii and B. afzelii strains. A 1271 bp DNA fragment of the 16 kb plasmid was cloned. It hybridized to several plasmids of this species (16, 27 and 55 kb). Sequencing of the cloned insert revealed a 327 bp ORF coding for a 14 kDa protein of unknown function, which could be expressed in E. coli. This ORF, conserved among B. burgdorferi sensu stricto strains, was carried by the same three plasmids.

Evidence for the involvement of different genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium.

In addition ot Borrelia burgdorferi, recognized as the aetiological agent of Lyme disease, at least two separate genospecies have recently been described. A relationship between infection by strains belonging to different genospecies and clinical outcome has been suspected. In this paper, 9 cases of Lyme arthritis were attributed to infection by B. burgdorferi sensu stricto, 18 cases of neuroborreliosis to B. garinii and one case of acrodermatitis chronica atrophicans to a strain of B. afzelii. These conclusions were based on the preferential reactivity of sera with antigens of given strains in Western blots and on residual reactivity after absorption of sera with antigens of representative strains. No conclusion could be reached concerning sera of 10 patients with erythema migrans.

Expression in Bacillus subtilis of the glycosomal glyceraldehyde-phosphate dehydrogenase gene from Trypanosoma brucei.

The cloned T brucei GAPDH gene was inserted within the B subtilis GAPDH gene, carried by pUC18. Upon transformation of B subtilis by this plasmid, not able to replicate in this host, the whole plasmid was inserted in the resident chromosome, presumably by a single recombination event between homologous, chromosomal and plasmid-borne sequences. The heterologous gene was expressed, as revealed by immunological reaction with monoclonal antibodies, recognizing specifically T brucei GAPDH. T brucei GAPDH, having little or no enzyme activity, comprises about 1.56% of cellular proteins. Peptide mapping showed that a fusion of a 7.5-kDa peptide had occurred to the N-terminal part of T brucei GAPDH. This fused protein is presumably the N-terminal part of B subtilis GAPDH, in agreement with the construction of the integrative plasmid.

Replication cycle of Bacillus subtilis hydroxymethyluracil-containing phages.

The present review focuses on phage 2C, a member of a family of virulent phages that multiply in Bacillus subtilis. The best known members of this group are SPO1, phi e, H1, 2C, SP8, and SP82, the genomes of which are made of double-stranded DNA of about 150 kilobase pairs (kbp). The two DNA strands have different buoyant densities. Moreover, thymine (T) is completely replaced by hydroxymethyluracil (hmUra). Comparison of the phage DNAs has shown that both base substitutions and deletions have contributed to the evolution of their genomes. In addition, all of the hmUra-phage genomes contain colinear redundant ends, amounting to 10% of total bases. Two lines of evidence suggest that the redundant ends of 2C DNA, in spite of extensive homology, contain unique sequences. Further studies focused on DNA replication during the lytic cycle. The semiconservative replication of the infecting viral genome is followed by extensive recombination. At the level of replication forks, viral DNA synthesis is discontinuous on both strands during the whole cycle. Deoxythymidinetriphosphate, required for viral DNA synthesis in permeabilized infected bacteria, was incorporated in small amounts into phage DNA. The putative primary origin of replication has been cloned and localized on the viral genome. Some viral promoters have been successfully cloned in Escherichia coli. These sequences, however, did not promote transcription in B. subtilis. The abnormal base might be required for promoter activity in the natural host.

Fluorometric in vivo determination of Bacillus subtilis and phage 2C DNA.

The validity of in vivo fluorometric assays was ascertained for phage and bacterial DNA measurements. The following parameters were determined by this simple technique. The DNA content of dividing cells of Bacillus subtilis 168/2 was 2.65 times higher than in resting cells. Assuming that resting cells harbor 1 genomic equivalent, its Mr was estimated to be 4.4 x 10(9) Da. A polymerization rate during growth of 788,000 bp min-1/cell is accounted for by a multifork replication mechanism. Both phage and host DNA could be measured accurately during the lytic cycle. Phage 2C DNA synthesis proceeded at a linear rate of 5.2 genome equivalents min-1.

Spectrofluorometry of dyes with DNAs of different base composition and conformation.

The increase in fluorescence, upon interaction with several fluorescent dyes was found to depend on the base composition of DNA. 4',6-Diamidino-2-phenylindole-2 HCl and Hoechst 33258 which bind to AT base pairs show a logarithmic relation. This relation is linear when DNAs interact with mithramycin, chromomycin A3, and olivomycin, which bind to GC base pairs. Deviations from these relationships were observed for T2 DNA, containing hydroxymethylcytosine, and for 2C DNA, containing hydroxymethyluracil. On the basis of these data, a simple technique is proposed for determination of base composition. The presence of abnormal bases can be monitored by the use of given fluorophores. Fluorescence intensities were not modified upon linearization of covalently closed circular plasmid pBR322. Denaturation of lambda DNA was accompanied by a decrease of fluorescence, when complexed with the five dyes tested.

Restriction analysis and quantitative estimation of methylated bases of filamentous and unicellular cyanobacterial DNAs.

The DNAs of strains of three cyanobacterial genera (Anabaena, Plectonema, and Synechococcus) were found to be partially or fully resistant to many restriction endonucleases. This could be due to the absence of specific sequences or to modifications, rendering given sequences resistant to cleavage. The latter explanation is substantiated by the content of N6-methyladenine and 5-methylcytosine in these genomes, which is high in comparison with that in other bacterial genomes. dcm- and dam-like methylases are present in the three strains (based on the restriction patterns obtained with the appropriate isoschizomeric enzymes). Their contribution to the overall content of methyladenine and methylcytosine in the genomes was calculated. Partial methylation of GATC sequences was observed in Anabaena DNA. In addition, the GATC methylation patterns might not have been random in the three cyanobacterial DNA preparations, as revealed by the appearance of discrete fragments (possibly of plasmid origin) withstanding cleavage by DpnI (which requires the presence of methyladenine in the GATC sequence).

Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis.

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.